Urothelium, also known as transitional epithelium, is a multi-layered epithelium covering a large part of the urinary tract including the renal pelvis, ureter, bladder and urethra. The apical surface of the urothelium is covered with numerous plaques of asymmetrical unit membranes whose luminal leaflet is almost twice as thick as the cytoplasmic leaflet. These rigid-looking plaques are believed to be involved in strengthening and stabilizing the apical surface during bladder distention, presumably through their interactions with an underlying cytoplasmic network of keratin filaments. Similar plaques are also associated with numerous cytoplasmic vesicles which are thought to be able to incorporate reversibly into the apical surface during bladder distention in order to satisfy the need for an increased cell surface area. EM studies shoved that the apical surface of these plaques is composed of semicrystalline arrays of hexagonal particles. During the last grant period we have established that these urothelial plaques contain three major proteins--a 27kD uroplakin I, a 15kD uroplakin II, and a 47kD uroplakin III. These three proteins are all urothelial-specific, are limited to the superficial umbrella cells, and are all AUK-associated in situ. Therefore, they represent excellent markers for an advanced stage of urothelial differentiation. In this next grant period, we will further characterize these three AUM-associated proteins by: 1) studying the interactions among the three uroplakins by chemical crosslinking, EM localization and scanning transmission electron microscopy; 2) defining the topology of uroplakins using proteases and peptide-specific antibodies; 3) studying the biosynthesis and processing of uroplakins; and 4) determining the molecular basis of urothelium-specific expression of uroplakin by gone cloning.